We have made NIH/3T3 cells tumorigenic by treatment with the ultimate carcinogen of benzo(a)pyrene, namely r-7, t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-benzo(a)-pyrene (anti-BPDE) and passaging in culture. Using these cells and mouse embryo fibroblasts (MEF) we propose to examine by somatic cell genetic approaches (1) suppression of tumorigenicity by fusion of cytoplasts of MEF or untreated NIH/3T3 cells to karyoplasts of BPDE-treated NIH/3T3 cells; (2) suppression of tumorigenicity of potentially transformed cells by fusion of cytoplasts of MEF or NIH/3T3 cells to karyoplasts of BPDE-NIH/3T3 cells which are not tumorigenic at passage 5; (3) induction of immortality of MEF by fusion of their karyoplasts to cytoplasts of BPDE-treated NIH/3T3 cells; and (4) induction of tumorigenicity by fusing cytoplasts derived BPDE-treated NIH/3T3 cells to karyoplasts of untreated NIH/3T3 cells. We have chosen to work with BPDE since it binds to mitochondrial DNA with a 50-100 times greater affinity than to nuclear DNA and produces tumors in rats and mice. Because many tumor cells have altered mitochondrial function, we will examine mitochondrial DNA for BPDE-induced mutations. Since it has been shown that BPDE introduces a specific mutation in the ras-Hal oncogene at codons 11 and 12 leading to a loss of an Mspl restriction site, we will initially analyze for loss of an Mspl restriction site in mitochondrial DNA obtained from BPDE-induced tumorigenic cells. To increase the probability of detection of such a mutation, mitochondrial DNA from tumorigenic BPDE-treated NIH/3T3 cells and nontumorigenic NIH/3T3 cells will be cloned. By examining which of the mitochondrial DNA eleven Mspl sites have been eliminated, we will be able to pinpoint the mutated gene(s).